The overall objective is to understand the mechanisms that regulate expression of the smooth muscle (SM) alpha-actin gene in smooth muscle cells (SMC) and to identify SM specific transcription factor(s). Identification of the regulators of SMC specific genes, such as SM alpha- actin, in SMCs is likely to be critical to understanding differentiation of SMC as well as alterations in differentiated phenotype that occurs in intimal SMC within atherosclerotic lesions. A key to understanding the altered expression of cell specific proteins is to identify mechanisms controlling the expression of genes that encode these proteins. Initial studies will focus on two regions of the SM alpha-actin gene promoter that have been shown in our previous studies to be involved in transcriptional regulation of the SM alpha-actin gene. To determine the importance of putative cis-elements in transcriptional regulation of the rat SM alpha-actin gene, wild-type and specific mutations of the rat SM alpha-actin promoter/reporter gene constructs will be transfected into rat aortic SMC and the activity of the reporter will be assayed. DNase I protection assays will be performed to determine the specific site(s) where rat aortic SMC nuclear factor(s) are binding. These factor(s) will then be identified by Southwestern cloning. Further studies will focus on identifying putative dimerization partners as well as the role of Id (a factor involved in regulating skeletal muscle differentiation) in SMC. These studies will include decreasing Id expression in SMC utilizing antisense oligonucleotides, and overexpressing Id by transfecting SMC with an expression plasmid containing the Id cDNA. Effects on SM alpha- actin protein synthesis will be assayed by 2D gel electrophoresis. Additionally, a two-hybrid system will be utilized to identify and subsequently clone genes that encode proteins that interact with Id in SMC.